Characterising the inflammasome response in a zebrafish model of haemorrhagic stroke

脑出血（ICH）是一种由脑内自发性出血引起的中风。血液毒性流入大脑引发炎症反应，诱导小胶质细胞活化、外周单核细胞和中性粒细胞浸润以及炎性细胞因子的产生。炎性小体是在炎症细胞中响应病原性感染和组织损伤而形成的胞质多聚体蛋白复合物。最近的研究强调了炎性小体激活在ICH后急性脑损伤进展中的重要作用。事实上，几项研究表明，在ICH实验模型中抑制炎性小体活化是有益的，并可改善结局（Ren et al，2018），开发新型炎性小体抑制剂用于治疗ICH后的急性脑损伤是最有前途的治疗途径之一。通过利用高分辨率实时成像并利用一系列在特定细胞/组织类型（例如血液、血管、垂死细胞、炎症细胞）中表达荧光蛋白的转基因报告细胞系，我们最近表征了脑出血斑马鱼幼虫模型中大脑中血液的病理后果（克里利et al，2018;克里利et al，2019）。我们发现，脑出血在斑马鱼幼虫引起脑细胞死亡的显着增加，增加神经炎症，并在运动功能的损害，从而模仿方面伴随人类脑出血的损伤表型。此外，我们已经证明该模型代表了一个强大的药物筛选平台，以鉴定减少出血后脑损伤的化合物（克里利等人，2022）。我们研究的下一个目标是利用ICH的斑马鱼模型筛选我们在实验室平行研究中表征的新型炎性小体抑制剂。尽管炎性小体通路的关键组分在功能上是保守的（Forn-Cuni et al，2019），但迄今为止，尚无研究描述斑马鱼ICH后的炎性小体反应。因此，该博士项目的目的是使用遗传学（CRISPR-Cas9），药理学（新型和现有的炎性小体抑制剂）和实时成像（转基因斑马鱼报告细胞系的光片/共聚焦显微镜）的组合来验证炎性小体激活在ICH后脑损伤进展中的作用。预期的项目成果是开发斑马鱼作为一种新工具，以i）询问炎性小体相关机制，ii）测试新的炎性小体抑制剂用于治疗ICH后的脑损伤。

Intracerebral haemorrhage (ICH) is a type of stroke caused by spontaneous bleeding within the brain. The toxic influx of blood into the brain initiates an inflammatory response that induces activation of microglia, infiltration of peripheral monocytes and neutrophils, and the production of inflammatory cytokines. Inflammasomes are cytosolic multimeric protein complexes formed in inflammatory cells in response to pathogenic infection and tissue injury. Recent research highlights an important role of inflammasome activation in progression of acute brain injury after ICH. Indeed, several studies indicate that inhibition of inflammasome activation in experimental models of ICH is beneficial and improves outcomes (Ren et al, 2018) and developing novel inflammasome inhibitors for the treatment of acute brain injury after ICH represents one of the most promising therapeutic avenues. By taking advantage of high-resolution live imaging and utilising a range of transgenic reporter lines that express fluorescent proteins in specific cell/tissue types (e.g. blood, blood vessels, dying cells, inflammatory cells), we have recently characterised the pathological consequences of blood in the brain in zebrafish larval models of ICH (Crilly et al, 2018; Crilly et al, 2019). We discovered that ICH in zebrafish larvae caused a significant increase in brain cell death, increased neuroinflammation, and an impairment in locomotor function, thus mimicking aspects of the injury phenotypes that accompany human ICH. Furthermore, we have shown this model represents a powerful platform for drug screening to identify compounds that reduce brain injury after haemorrhage (Crilly et al, 2022).The next aim of our research is to utilise the zebrafish model of ICH for screening novel inflammasome inhibitors that we are characterising in the lab in parallel studies. Although key components of the inflammasome pathways are functionally conserved (Forn-Cuni et al, 2019), to date, no studies have described the inflammasome response after ICH in zebrafish. Therefore, the aim of this PhD project is to use a combination of genetic (CRISPR-Cas9), pharmacological (novel and existing inflammasome inhibitors) and live imaging (light sheet / confocal microscopy of transgenic zebrafish reporter lines) to characterise the role of inflammasome activation in the progression of brain injury after ICH. The intended project outcome is the development of zebrafish as a new tool to i) interrogate inflammasome-related mechanisms and ii) test new inflammasome inhibitors for the treatment of brain injury after ICH.